The present invention relates to the field of diagnosing infections of Pseudomonas aeruginosa through the analysis of characteristic volatile metabolites associated with such infections.
Pseudomonas aeruginosa is an opportunistic pathogen which is responsible for serious skin infections in burn patients and for debilitating lung infections in patients with cystic fibrosis. This pathogen is also the cause of many wound and urinary tract infections, and accounts for about 15% of all hospital-acquired infections. Early detection of the nature and extent of such infections is important to the overall treatment of infected patients.
Several approaches have previously been suggested for determining the nature and extent of Pseudomonas aeruginosa infections in subject patients. It has been suggested to culture the Pseudomonas aeruginosa for extended periods of time (between 24-48 hours) on a suitable medium, and ultimately to subject the cultures to various chemical tests and morphologic examinations. The chemical tests are based upon the fact that each organism has unique metabolic capabilities ie., sugar fermentation and oxidation, amino acid decarboxylase and dihydrolase production, H.sub.2 S generation, etc. Unfortunately, such methods are slow and laborious, and may not provide diagnostic indications which are prompt enough to facilitate optimum treatment of the infection.
It has also been suggested to detect the fluorescence of various Pseudomonas by-products on the skin of burn patients in an attempt to quantify the nature and extent of Pseudomonas aeruginosa infections. Quantification of the extent of such infections through this method is, of course, difficult, and at present, such methods have not been successfully adapted for use in diagnosing the nature and extent of such infections in cystic fibrosis patients.
Various investigators have suggested that Pseudomonas aeruginosa may be identified through its secondary metabolites. It has been suggested, for example, that various Pseudomonas species including Pseudomonas aeruginosa may be identified through various cellular fatty acids produced by such species. See for example, Moss, C. W. and S. B. Dees, 1976, "Cellular fatty acids and metabolic products of Pseudomonas species obtained from clinical specimens"; Journal of Clinical Microbiology 4: 492-502. It has also been suggested that methyl esters of such fatty acids may be identified using gas chromatographic characterization procedures. See Wade, T. J. and R. J. Mandel, 1974, "New gas chromatographic characterization procedure: preliminary studies on some Pseudomonas species", Applied Microbiology 27: 303-311.
It has further been indicated that 2-amioacetophenone may be a useful indicator of Pseudomonas aeruginosa when cultures of Pseudomonas aeruginosa are subjected to an ether extraction of the culture and a subsequent GC/MS analysis. The results of such analysis are then compared to a known profile of 2-aminoacetophenone to determine the nature and extent of any Pseudomonas aeruginosa infection. See "Use of 2-aminoacetophenone production in identification of Pseudomonas aeruginosa", by Charles D. Cox and J. Parker, Journal of Clinical Microbiology, Vol. 9, No. 4, pgs. 479-484 (April, 1979). Similarly, in media supplemented with methionine, Pseudomonas aeruginosa has been reported as producing dimethyldisulfide, but not methyl mercaptan. See "Development of specific tests for rapid detection of E. coli and species of Proteus in urine", by N. J. Hayward, et al, Journal of Clinical Microbiology, Vol. 6, No. 3, pgs. 195-201 (September, 1977). Other species of Pseudomonas, such as P. putida, P. fluorescens, and P. putrefaciens have variously been reported as showing the presence of 2-nonanone, dimethyldisulfide, dimethyltrisulfide, and other sulfur metabolites. See "Volatile Compounds Produced in Sterile Fish Muscle (Sebastes malanops) by Pseudomonas putrefaciens, Pseudomonas fluorescens, and an Achromobacter Species", by Miller et al, Applied Microbiology, Vol. 26, No. 1, pgs. 18-21 (July, 1973). See also "High resolution gas chromatographic profiles of volatile organic compounds produced by microorganisms at refrigerated temperatures", by Lee et al, Applied and Environmental Microbiology, Vol. 37, No. 1, pgs. 8590 (January, 1979).
Other literature of particular interest in this area includes articles entitled "Epidemiology of Pseudomonas aeruginosa infections: determination by pyocin typing", by Bruun et al, (1976) Journal of Clinical Microbiology, 3:264-271; and "Pseudomonas carrier rates of patients with cystic fibrosis and of members of their families", by Laraya-Cuasay et al, Journal of Pediatrics, 89:23-26 (1980).
Recently, the application of gas chromatography to the indentification of unknown microorganisms has received wide-spread attention. See "Gas chromatography application in Microbiology and medicine", by Mitruka (1979), John Wiley and Sons, New York. The techniques which have been developed are based on analysis of either the unique metabolites of a given organism or on its individual structural components. Culture extracts have, for example, revealed specific amines for Clostridia (Brooks et al), "Further studies on the differentiation of Clostridium sordelli Clostridium bifermentans by gas chromatography", (1970) Can. J. Microbiol. 16: 1071-8. Specific hydroxy acids and fatty acids have been identified for Neisseria. Brooks et al, "Analysis by gas chromatography of hydroxy acids produced by several species of Neisseria", Can. J. Microbiol. 18: 157-168 (1972); Brooks et al, "Analysis by gas chromatography of fatty acids found in whole cultural extracts of Neisseria species", Can. J. Microbiol. 17: 531-541. As mentioned above, bacteria cellwall preparations have been examined for unique fatty acid profiles, including such profiles for Pseudomonads. Moss, supra. (1976); and Wade, supra. (1974). Pyrolysis-gas chromatography of whole cell Clostridia bacteria has also been reported as giving identifiable differences in the observed fragmentation patterns. Reiner, et al, "Botulism: A pyrolysis-gas-liquid chromatographic study", J. Chromatogr. Sci. 16: 623-629 (1978).
Headspace analysis techniques have also been developed to sample directly the volatile metabolites produced in culture. These have involved either sampling the culture headspace directly, as in the case of aliphatic acids and amines for various anaerobes, and sulfides for proteus; or have made use of volatile concentration methods such as for Pityrosporum. For literature reporting on such techniques, please refer to Bohannon et al, "Quantitative methods for the gas chromatographic characterization of acidic fermentation by-products of anaerobic bacteria", J. of Chromatogr. Sci. 16: 28-35 (1978); Larsson, et al, "Analysis of amines and other bacterial products by head-space gas chromatography", Acta Path. Microbiol. Scand. Sect B 86: 207-213; Hayward, et al, "Methylmercaptan and DMDS production from methionine by Proteus species detected by head-space gas liquid chromaography", J. of Clin. Microbiol 6: 187-94 (1977). See also Labows, et al, "Characteristic Gamma-Lactone ordor production of the genus Pityrosporum", Appl. and Environ. Micro 38: 412-415 (1979); Lee, et al, supra. (1979); and Morgan, "The chemistry and some microbially-induced flavor defects in milk and dairy foods", Biotech. Bioeng. 18: 953-965 (1976). Headspace analysis has also been applied to samples of human body fluids including salvia, urine and blood serum. For references on this topic, please refer to Kostelc, et al, "Salivary volatiles as indicators of periodontitis", J. Periodont. Res. 18: 185-192 ( 1980); Matsumota, et al, "Indentification of volatile compounds in human urine", J. Chromatogr. 85: 31-34 (1973); Zlatkis, et al, "Concentration and analysis of volatile urinary metabolites", J. Chromatogr. Sci. 11: 299-302 (1973); Liebich, et al, "Volatile substances in blood serum: profile analysis and quantitative determination", J. Chromatogr. 142: 505-516 (1977).
It has further been suggested to manipulate the production of secondary metabolite production in Pseudomonas through the systematic optimization of medium composition or growth conditions, or by mutation. For example, it has been suggested to alter pyocyanine production by providing a selected supply of Fe.sup.2+, or altered amounts of phosphates. See Leisinger, et al, supra. at 435-436.